The ideal, most effective, tool for eliminating Plasmodium falciparum (Pf), the causative agent of 99% of all malaria deaths, is a highly effective vaccine that prevents blood stage infection and thereby prevents both disease and transmission. Sanaria's goal is to develop and commercialize a Pf sporozoite (SPZ) vaccine that prevents Pf blood stage infection in >90% of recipients. The Sanaria(R) PfSPZ Vaccine is composed of attenuated, purified, aseptic, cryopreserved PfSPZ. The platform technology developed to manufacture the vaccine has also facilitated the manufacture of PfSPZ for challenge infections to test vaccines and drugs (PfSPZ Challenge) and for vaccination by challenge under chemoprophylaxis (PfSPZ-CVac). These comprise a portfolio of products that are on an aggressive timeline to commercialization. Manufacture of all these PfSPZ products is dependent on Anopheles stephensi mosquitoes that can survive aseptic rearing from eggs to adults and produce high numbers (>105) of PfSPZ in their salivary glands. A vulnerable aspect of Sanaria's manufacturing process is its reliance on mosquitoes that can only be maintained in colonies; such colonies are susceptible to crashes and to genetic drift over time, thereby being at risk of permanently losing the desired genotype and phenotype. This is because despite decades of effort, until our breakthrough achievement in Phase I, live Anopheles could not be preserved in any way. In Phase I we demonstrated new enabling technology, the capability to cryopreserve eggs of A. stephensi (SDA500-9800 strain). In the GMP context, being able to cryopreserve large lots of eggs will allow Sanaria to inaugurate controlled manufacturing by use of a Master Mosquito Bank, thereby enabling us to return repeatedly to a repository stock generated from the same defined, characterized material with the desired genetic background. With careful management, such a stock can last for many tens of years. In this Phase II project we will fully optimize the cryopreservation method (Specific Aim 1), and use this method in scaled-up production, initially to ensure security of the colony for manufacturing purposes (Specific Aim 2) and subsequently for producing the larger Master Mosquito Egg Bank (Specific Aim 3) from which all mosquitoes used in manufacturing will be sourced. Details of the characterized Master Mosquito Egg Bank will be submitted to the FDA as a Biologics Master File. We will also systematically apply the egg cryopreservation technology to support development of improved strains of A. stephensi and to create banks of eggs of such strains when developed (Specific Aim 4). Success in this project will have a critically important impact on Sanaria's commercial success.